Gen5

Cells were plated in 96-well plates at 0.5 × 10⁵ cells per well (24 h toxicity assay) and 0.04 × 10⁵ cells per well (7 day proliferation study). After experiment endpoints, resazurin (Alamar Blue) (Millipore-Sigma, St. Louis, MO, USA) indicator dye was used to measure viable cell count. Briefly, a working solution of resazurin (0.5 mg/mL) was prepared in sterile PBS; filter sterilized through a 0.2 micron filter; added to the samples (15% (v/v) equivalent); and returned to the incubator for 2–6 h. Reduction of the dye by viable cells reduced the oxidized resazurin, yielding a bright red fluorescent intermediate resorufin, quantified using a Synergy multi-mode reader (Model HTX, Bio-Tek (Agilent), Winooski, VT, USA) using 530 nm (excitation)/590 nm (emission) filters. Spheroids were grown in the ultra-low attachment (ULA) 96-well plates, which enable the self-assembly of spheroids grown to maturation for 7 days achieving uniformity in size and shape. After maturation, GNE-140 was added to the wells, and spheroids were grown for another 14 days followed by image analysis using phase contrast and fluorescence digital microscope photography using a Biotek Cytation 5 imaging Station and Gen 5.0 software (Winooski, VT, USA).

iCMs were stained for senescence-associated β-galactosidase (SA-β-gal) activity as previously described 35 (link). Briefly, cells were washed twice with PBS, fixed with 2% formaldehyde and 0.2% glutaraldehyde in PBS, and washed twice in PBS. Cells were stained in X-gal staining solution (1 mg/ml X-gal, 40 mM/l citric acid/sodium phosphate, 5 mM/l potassium ferricyanide, 5 mmol/l potassium ferrocyanide, 150 mM/l NaCl, 2 mM/l MgCl₂, pH 6.0). After 6 hours, cells were washed twice with PBS. For a sensitive determination of the total cell number, cells were counterstained with 1 µg/ml Hoechst 33342 (Molecular Probes). Stained cells were examined using Lionheart FX Automated Microscope (BioTek Instruments Inc., Winooski, VT, USA) and analyzed with Gen 5.0 software (Biotek Instruments).

Competitive binding of inhibitors to HSP90α was monitored using FITC-GM in a FP assay [47 (link)]. Briefly, compound dilutions were incubated with HSP90α (90 nM final) and FITC-GM (2 nM final) in a 96-well microplate at 4°C for 16 h in the presence of assay buffer (20 mM HEPES, pH 7.4, 50 mM KCl, 5 mM MgCl₂, 20 mM NaMoO₄, 0.01% NP40, 2 mM DTT and 0.1 mg/ml BSA). Polarization was measured in Synergy H4 Hybrid reader (BioTek, Winooski, VT, USA) using Gen5.0 software (BioTek). [49 (link)]

Cellular viability was assessed by double labeling of cells with 1 μM calcein-AM and 1.2 μM DRAQ7; total nuclei were stained with 1µg/ml Hoechst 33342 (Merck). Viable total and dead cells were counted using the Lionheart FX Automated Microscope (BioTek Instruments Inc., Winooski, VT, USA). Apoptosis was further evaluated by Terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay. CMs were treated and after 24hours fixed in 4%methanol-free Paraformaldehyde (PFA) for 15 min then processed with DEadEnd Fluorimetric TUNEL system (Promega) following manufacturer protocol. Total cells were stained with DAPI 1µg/ml (Merck).
Stained cells were acquired using Lionheart FX Automated Microscope (BioTek Instruments) and analyzed with Gen 5.0 software (BioTek Instruments).

The WM164 cells were cultured for the formation of spheroids in DMEM containing 20 ng/mL epidermal growth factor, 10 ng/mL basal fibroblast growth factor, 5 µg/mL insulin and 0.4% bovine serum. Cells at a concentration of 5 × 10³/mL were added to the medium and incubated with factor B27, growth factor to support the formation of spheroids in cell lines, at a dilution of 1:50 (Gibco, Waltham, MA, USA). The cells were plated onto a 96 well ultralow attachment plate (Costar^®, Corning, NY, USA) 200 µL/well. The wells at the edge of the plate were filled with PBS to ensure adequate humidity inside the plate. After a week of incubation, the resulting spheroids were counted manually and using a Cytation 5 instrument (BioTek, Winooski, VT, USA). Data were analyzed using Gen 5.3 software (BioTek, Winooski, VT, USA).